Metal ions in solution are attracted to the vicinity of functionalized polymer molecules. The reduction of the metal ions to metal(O) atoms creates a condition of super-saturation with respect to metal(O) atoms at a given polymer site. Consequently, the nucleation of metal particles proceeds in the local domain of the individual polymer molecules. Steric repulsion between different polymer-metal domains keeps the domains separated while attractive force between the polymer's functional groups and metal atoms on a particle keeps the particle in a single domain. The net effect is the immobilization of metallic nuclei by individual polymer molecules and the prevention of metal(O) particle growth by coagulation, i.e., nuclei from different polymer molecules are prevented from migrating and coagulating to form larger particles.
While polymer molecules may prevent metal(O) particle growth by coagulation, particle growth can occur by the diffusion of metal ions to the vicinity of the metal(O) particle nuclei. The growth of the particles is terminated when the solution is depleted of metal ions capable of migration. The metal(O) particles that are formed are uniform in size and shape. Further information on homogeneous precipitation, may be found in A. E. Nielsen, "Kinetics of Precipitation" (McMillan, New York 1964); E. Matijevic, Accts. Chem. Res., 14: 22 (1981); and T. Sugimoto, Adv. Coll. Interface Sci., 25: 65-108 (1987).
Colloidal dispersions of different metals have been prepared through a judicious choice of polymer, metal ion, reducing agent, the selective concentrations of the various ingredients and the temperature at which the reactions are carried out. Simple alcohols such as methanol or ethanol have been used as both solvent and reductant in the presence of a protecting polymer such as PVA (polyvinyl acetate). Polyols, ethylene glycol and diethylene glycol have been used similarly as both solvent and reductant. Functionalized polymers such as hydrazinium polyacrylate have been used as both reductant and protecting polymer in the preparation of monodispersed particles of gold, silver, copper, platinum and selenium. Tannin, which contains one to three trihydroxybenzenecarboxylate groups on a sugar, usually glucose, can act as both a reducing agent and a protecting substance during the formation of gold hydrosols. For examples, see L. Hernandez et al, J. Colloid Sci., 3: 363-375 (1948) (rhodium-polyvinyl acetate [Rh-PVA] polymer using hydrogen as reductant); H. Thiele et al, J. Colloid Sci., 20: 679-695 (1965) (hydrizide of polyacrylic acid (PAH)-Au, Ag, Cu, Pt and polyethyleneimi acid-Au); H. Harai et al, J. Macromol. Sci. Chem., A12: 1117-1141 (1978) (Rh-PVA using methanol as reductant); H. Harai et al., J. Macromol. Sci. Chem., A13: 633-649 (1979) (Rh-, Pd-, Os-, Ir- and Pt- PVA using methanol as reductant); H. Harai et al, ibid., A13: 727-750 (1979) Rh-, Pd-, Ag-, Os-, and Ir-PVP using ethanol as reductant); F. Fievet et al, MRS Bulletin, December, 1989, pp. 29-34 (Au-, Ag-, Pd-, Pt-, Cu-, Co-, Ni-, Cd- and Pb-Polyol); O. Siiman et al., J. Chem. Soc. Faraday Trans., 82: 851-867 (1986) (Au-PVP-ethanol); H. B. Wieser, "Inorganic Colloid Chemistry, Vol. I, The Colloid Elements" (Wiley, New York (1933)) (tannin-Au); T. W. Smith et al., Macromol., 13: 1203-1207 (1980) (Se-PA hydrazine) and T. W. Smith et al., J. Phys. Chem., 84: 1621-1629 (1980) (Fe-polymer).
Colloidal gold has been used frequently as an adsorbent for antibodies, enzymes, lectins and other proteins, primarily because of the non-specific nature of protein adsorption on colloidal gold. Virtually any protein material may be adsorbed onto colloidal gold. Protein-gold species, maintained in an active state, have found use as cell surface markers in optical and electron microscopy. [See "Colloid Gold: Principles, Methods and Applications", Vols. I, II and III, M. A. Hayat, ed. (Academic Press, New York 1989) and A. J. Verkleij and J. L. M. Leunissen, "Immuno-Gold Labelling In Cell Biology" (CRC Press, Boca Raton, Fla. 1989)]. The detection of colloidal metal particles of 10-100 nm diameter in light microscopy by epi-illumination with polarized light is described in U.S. Pat. No. 4,752,567 to M. J. De Brabander et al. Of particular interest are colloidal gold particles having directly or indirectly adsorbed antibodies. Indirect adsorption is accomplished by binding the antibody to a first adsorbed layer; for example, a secondary antibody, avidin, or protein A or G. Ligand-dextran complexes have been adsorbed onto colloidal gold. The ligands included lectins, concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin [D. Hicks and R. S. Molday, Invest. Opthalmol. Vis. Sci., 26: 1002-1013 (1985)] and ouabain [R. J. Mrsny et al., Eur. J. Cell Biol., 45: 200-208 (1987)].
However, the adsorption of proteins onto colloidal gold particles in aqueous solution is a reversible equilibrium reaction. Particle adsorbed protein and unabsorbed or free protein are separated, usually by centrifugation. However, after separation and removal of the free protein, and resuspension of the purified protein-gold colloid, further loss of adsorbed material occurs as equilibrium re-establishes itself. Consequently, long term storage of purified adsorbed protein-gold conjugates has not been practical. Some workers in the field have suspended the purified protein-gold colloids in buffer solutions containing blocking agents such as bovine serum albumin (BSA), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) and other polymeric materials in an attempt to increase stability. The polymeric materials serve to cover any remaining exposed areas on the surface of the colloidal gold particles and thus prevent aggregation of gold particles. However, the polymeric material is present in large excess over the adsorbed protein on the colloidal gold and competes with the protein for colloidal gold sites. Inevitably, some desorption of protein from gold will occur and free protein will be present again in the solution.
It is an object of the invention to prepare colloidal metal particles, preferably colloidal gold or silver particles, having a stabilized coating of a polysaccharide material. Another object of the invention is to use such coated colloids to prepare stable protein-colloid conjugates. The protein-colloid conjugates of the invention overcome the instability and orientation problems associated with proteins which are adsorbed directly on colloid metal surfaces.